The structure and function of the apical aspect of polarized cells is determined by the cortical cytoskeleton that attaches to the apical plasma membrane. Previously a protein called ezrin was identified that is a specific cytoskeletal component of apical microvilli. Ezrin is the founding member of the ERM (ezrin-radixin-moesin) family of proteins, which are involved in linking actin filaments to the plasma membrane. Work accomplished during the current period has revealed that ezrin is a conformationally regulated protein, so that binding sites for actin and membrane association are masked due to an intramolecular association between two domains called the N- and C-ERMADs (ERM association domains). Activation of ezrin leads to exposure of the N- and C- ERMADs which results in ezrin oligomer formation as well as exposure of sites for binding F-actin and membrane components. A protein, EBP50, was identified that binds ezrin and has two PDZ domains that are predicted to interact with the C-terminal tails of membrane proteins. The cytoplasmic tail of the cystic fibrosis transmembrane conductance regulator (CFTR) was found to interact with PDZ1 of EBP50. Based on these results, we propose that ezrin provides a regulated link from the actin cytoskeleton, through EBP50 to membrane proteins. It is proposed to test many aspects of this model. First, based on the three dimensional structure of the N- and C-ERMAD complex that we shall soon have, we shall make site-directed mutations in ezrin that should constitutively activate the molecule; the biochemical and physiological consequences of these changes will be evaluated. Second, biochemical methods will be used to identify and characterize proteins that bind activated, but not dormant, ezrin. Third, transfection experiments will be used to determine the role of EBP50 in the organization of cell surface structures. The hypothesis that EBP50 tethers membrane proteins to the plasma membrane and restricts them from entering the endocytic cycle will be examined. Fourth, biochemical approaches will be used to determine the repertoire of proteins that bind the PDZ domains of EBP50. Fifth, the mechanism of ezrin activation will be investigated. Sixth, in a collaborative study, we shall extend our structural studies to examine ezrin bound to other ligands (e.g. EBP50) as well as different conformational states of ezrin. These studies will provide a comprehensive analysis of ezrin and EBP50 and the role they play in the structure and function of the apical membrane.